CD4+CD25high regulatory T cells (Tregs) isolated from with the circulation of patients with a number of different autoimmune diseases, including type I diabetes, MS, and rheumatoid arthritis, exhibit deficient in vitro suppressive activity when compared to Tregs from healthy control subjects. These data suggest that loss of the CD4+CD25h'9h Treg function may in part underlie the pathophysiology of human autoimmune disease. Nevertheless, while it is likely that Tregs are critical for the regulation of autoimmunity, their mechanism of action and the sites where they function in human disease in unknown. Our recent preliminary data demonstrates that the population of CD4+CD25high Tregs contains two distinct subpopulations of that can be discriminated by their ex vivo expression of the MHC Class II molecule, HLA-DR: DR+CD4+CD25high (DR+ Treg) and DR~CD4+CD25high (DR~ Treg);specifically, DR+ Tregs enforce a rapid and complete suppression that is cell-contact dependent and mediated by cell surface expression of TGFIi, while DR Tregs promote Th2 cytokine production in responding T cells. This Project in the U19 grant will focus on: 1) understanding which subset (DR+ Treg or the Th2 inducing DR" Treg) is altered in patients with type I diabetes and MS, followed by examining the mechanism for the loss of suppressive function;2) determining whether the decrease in circulating CD4+CD25high Tregs is due to migration to the tissue site of inflammation and what is the mRNA expression profile of Tregs in the draining lymph nodes of human diabetics as compared to control;and 3) using a RNAi libraries and RNA expression arrays, we will interrogate the biochemical mechanisms associated with loss of function of the regulatory T cell populations in humans with diabetes and MS. Efforts to identify genes by whole genome RNAi libraries that when silenced allow gain of defective Treg function from patients with MS and diabetes will be explored. The overall aim of this U19 is to ultimately develop tools that can be used to manipulate regulatory T cell populations to restore function and prevent autoimmune disease.